Drug Testing in Cellular Chemotaxis Assays

Mario Mellado1, Carlos Martínez‐A1, José Miguel Rodríguez‐Frade1

1 Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, Madrid, Spain
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 12.11
DOI:  10.1002/0471141755.ph1211s41
Online Posting Date:  June, 2008
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Described in this unit are methods for measuring the cell migration process. While cell adhesion protocols allow study of migrating cell interactions with the endothelial matrix, cellular migration assays permit analysis of directed cell movement towards a chemotactic gradient, both in vivo and in vitro. An in vitro cell invasion protocol is provided for analysis of the sum of the cell adhesion, migration, and invasion activities involved in tumor cell motility. Curr. Protoc. Pharmacol. 41:12.11.1-12.11.22. © 2008 by John Wiley & Sons, Inc.

Keywords: adhesion; migration; chemokines; integrins; selectins

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Steady-State 96-Well Plate Cell Adhesion Assay
  • Alternate Protocol 1: Steady-State Cell Adhesion Assay on Slides: Drop Assay
  • Alternate Protocol 2: Cell Adhesion Under Flow Conditions
  • Basic Protocol 2: Transwell Migration Assay for Cells in Suspension
  • Alternate Protocol 3: Cell Migration Assay on Frame Filters for Adherent Cells
  • Alternate Protocol 4: Murine In Vivo Cell Migration Assay
  • Basic Protocol 3: Assessment of Compound Effects on In Vitro Tumor Cell Invasion
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Steady-State 96-Well Plate Cell Adhesion Assay

 Materials
  • Substrates (available from BD Biosciences, Sigma-Aldrich, R&D Systems, Calbiochem, Chemicon; store stocks solutions up to 1 year at –20°C), e.g.:
    • 150 mM fibronectin stock in PBS or other aqueous medium (e.g., tissue culture medium)
    • 5 mg/ml collagen stock in 20 mM acetic acid
    • 150 mM VCAM stock in PBS or other aqueous medium (e.g., tissue culture medium)
  • Phosphate-buffered saline (PBS; see recipe)
  • Chemokines (R&D Systems, Peprotech; resuspend to 10 µM in sterile PBS; store up to 6 months at –20°C in 5- to 10-µl aliquots; avoid multiple freeze-thaw cycles)
  • Test compound(s)
  • 2% (w/v) bovine serum albumin (BSA)
  • BCECF-AM [2¢,7¢-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester] (Calbiochem, Molecular Probes)
  • Dimethylsulfoxide (DMSO)
  • Target cells of interest, e.g.:
    • Adherent cell lines (HEK-293, 3T3 fibroblasts, MeWO, BLM)
    • Cells growing in suspension (Molt4, Jurkatt, Mono Mac-1, THP-1)
    • Primary cells (isolated human and mouse primary monocytes, B and T cells)
  • Depletion medium: serum-free DMEM or RPMI-1640 medium containing 0.5% (w/v) bovine serum albumin (BSA)
  • Tissue culture medium (DMEM or RPMI-1640)
  • PBS (see recipe) containing 0.5% (w/v) sodium dodecyl sulfate (SDS)
  • 96-well flat-bottom plates with high protein-capture capacity (e.g., Nunc, Costar, BD Falcon)
  • Orbital shaker (e.g., Belly Dancer, Stovall Life Science, http://www.slscience.com/)
  • Centrifuge with microtiter plate carrier
  • Fluorimeter (Millipore; Thermo Scientific; Tecan; BMG Labtech, http://www.bmglabtech.com)
  • Additional reagents and equipment for flow cytometry (Robinson et al., 2008)

Alternate Protocol 1: Steady-State Cell Adhesion Assay on Slides: Drop Assay

 Materials (also see Basic Protocol 1)
  • Fetal bovine serum (FBS)
  • Hanks] balanced salt solution (HBSS, e.g., Sigma, Invitrogen) containing 10 mM HEPES, pH 7.5
  • Hanks' balanced salt solution (HBSS, e.g., Sigma, Invitrogen) containing 10 mM HEPES, pH 7.5, and 1.5% (w/v) glutaraldehyde
  • 12-well chamber slides (Nunc)
  • Humidified chamber: e.g., covered plastic container with moistened paper towels on the bottom
  • 37°C heating block
  • Image J software (download free at http://rsb.info.nih.gov/ij/)

Alternate Protocol 2: Cell Adhesion Under Flow Conditions

 Additional Materials (also see Basic Protocol 1)
  • Substrate solution: 20 µg/ml fibronectin, 20 µg/ml collagen, or 4 µg/ml MadCAM (prepare from stock solutions described in Basic Protocol 1 materials list)
  • Tissue culture medium (DMEM or RPMI-1640) containing 10% (v/v) FBS
  • Depletion medium: serum-free DMEM or RPMI-1640 containing 2% (w/v) BSA
  • Tissue culture medium (DMEM or RPMI-1640) containing 50 µM EDTA
  • Tissue culture medium (DMEM or RPMI-1640)
  • PBS (see recipe) containing 0.05% (w/v) sodium azide (prepare from 10% w/v NaN3 stock in H2O stored up to 1 year or longer protected from light in amber glass at room temperature)
  • 100-mm petri dishes (e.g., Nunc, Falcon-BD Systems, Sigma-Aldrich)
  • Permanent marker
  • Parallel flow chamber (e.g., Glycotech, http://www.glycotech.com; see Fig. 12.11.2) with syringe pump (e.g., Harvard Apparatus) and vacuum pump
  • Inverted light microscope
  • CCD camera (e.g., Cohu, http://www.cohu.com)
     FigureFigure 12.11.2 Photograph and schematic model of the flow chamber.

Basic Protocol 2: Transwell Migration Assay for Cells in Suspension

 Materials
  • Substrate (optional): endothelial cells (HUVEC or bEnd mouse brain endothelial cells), 5 µg/ml fibronectin, or 10 µg/ml collagen (prepare from stock solutions described in Basic Protocol 1 materials list)
  • Depletion medium: serum-free DMEM or RPMI-1640 containing 2% (w/v) BSA
  • Chemottractants, chemokines, or test compounds of interest (resuspend chemokines to 10 µM in sterile PBS and store frozen at –20°C for up to 6 months in 5- to 10-µl aliquots to avoid repeated freeze-thaw cycles)
  • Migration medium: DMEM or RPMI 1640 containing 25 mM HEPES and 0.1% (w/v) BSA
  • 0.5% (w/v) crystal violet in 20% (v/v) methanol (store at room temperature up to 6 months; use for adherent cells)
  • Transwells, 3-, 5-, or 8-µm pore size (Costar, BD Biosystems, Nunc, Corning)
  • Centrifuge
  • Cotton swabs (for adherent cells)
  • Luminometer (Millipore; Thermo Scientific; Tecan; BMG Labtech, http://www.bmglabtech.com)

NOTE: Disposable 24-well transmigration chambers are recommended for cells growing in suspension. The pore size depends on cell type (see Critical Parameters). Sterile conditions are usually unnecessary because cell migration experiments are conducted over a brief period of time (less than 4 hr). If assay must be adapted for adherent cells, see step 5, below; also see Alternate Protocol 3.

Alternate Protocol 3: Cell Migration Assay on Frame Filters for Adherent Cells

 Materials
  • 20 µg/ml collagen VI or 5 µg/ml fibronectin (i.e., Sigma-Aldrich, BD Biosciences), prepared in distilled H2O
  • 5 mM tetrasodium EDTA
  • 0.05% (w/v) trypsin/5 mM tetrasodium EDTA
  • Migration medium: RPMI-1640 or DMEM containing 25 mM HEPES and 0.1% (w/v) BSA
  • Chemoattractant or agonist test compounds in migration medium
  • 0.5% (w/v) crystal violet in 20% (v/v) methanol (store up to 6 months at room temperature)
  • Permanent marker
  • Frame filters, 8- or 10-µm pore size (Neuro Probe Inc.)
  • Centrifuge
  • Surgical instruments, sutures, and wound clips or skin staples
  • Migration chamber (Neuro Probe Inc.)
  • Cell scraper
  • Densitometer

Alternate Protocol 4: Murine In Vivo Cell Migration Assay

 Materials
  • Cells of interest
  • Vital dye cell trackers (Molecular Probes, Calbiochem, Promega, Sigma-Aldrich), e.g.:
    • BCECF-AM (absorption/emission 475/535 nm)
    • CMTMR (541/565 nm)
    • CMFDA (492/517)
    • Calcein-AM (494/517 nm)
  • Dimethylsulfoxide (DMSO), anhydrous
  • Phosphate-buffered saline (PBS; see recipe)
  • Mice (C57BL/6, BALB/c; healthy animals of any age or gender)
  • Anesthetic (ketamine and xylazine; also see Donovan and Brown, 1998)
  • Chemoattractant or agonist test compounds
  • Chemokine (optional; resuspend to 10 µM in sterile PBS; store up to 6 months at –20°C in small aliquots; avoid repeated freeze-thaw cycles)
  • Tissue culture medium (DMEM or RPMI-1640)
  • Erythrocyte lysis buffer: 0.85% (w/v) NH4Cl in H2O
  • Centrifuge
  • 100-mm petri dish
  • 40-µm nylon cell strainers
  • 15-ml centrifuge tubes
  • Additional reagents and equipment for injection of mice (Donovan and Brown, 2006a), anesthesia of mice (Donovan and Brown, 1998), euthanasia of mice (Donovan and Brown, 2006b), survival surgery/splenectomy on mice (Reeves et al., 1991), and flow cytometry (Robinson et al., 2008)

Basic Protocol 3: Assessment of Compound Effects on In Vitro Tumor Cell Invasion

 Materials
  • Matrigel (BD Biosciences)
  • Cells: e.g., tumor cells, neutrophils, eosinophils or endothelial cells
  • Chemoattractants or other test compounds
  • Depletion medium: serum-free DMEM or RPMI-1640 containing 0.5% (w/v) BSA
  • 4% (w/v) paraformaldehyde in PBS (see recipe for PBS)
  • 0.1% (w/v) crystal violet in 20% (v/v) methanol
  • 24-well transmigration chambers (8-µm pore size; e.g., Costar, BD Biosystems, Nunc, Corning)
  • Cotton swabs
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  •  FigureFigure 12.11.1 (A) CXCL12-induced adhesion of human PBL to fibronectin. Cells were loaded with BCECF-AM, pretreated with the CXCR4 antagonist AMD3100 (50 ng/ml, 20 min 37°C) or control, and allowed to adhere to CXCL12 coimmobilized with fibronectin. (B) CCL2-induced adhesion of human purified monocytes to fibronectin. Cells loaded as in (A) were pretreated with mIgG2b (an antagonist anti-CCR2 monoclonal antibody), CCR2-05, or isotype-matched control (5 µg/ml, 30 min, 37°C). Cell adhesion to CCL2 coimmobilized with fibronectin was determined. Results are shown as mean fluorescence of quadruplicate determinations in a representative experiment.
    View Image
  •  FigureFigure 12.11.2 Photograph and schematic model of the flow chamber.
    View Image
  •  FigureFigure 12.11.3 Photograph and schematic of the flow chamber installation used to visualize and monitor cell tethering, rolling, and adhesion under flow conditions as described in Alternate Protocol 2.
    View Image
  •  FigureFigure 12.11.4 Alternate Protocol 2. CXCL12-promoted adhesion to fibronectin under flow conditions of human Molt 4 cells. (A) Cells, untreated or pertussis toxin (PTx)–treated, were infused in the flow chamber and analyzed for detachment at increasing flow rates. Cells are expressed as the percentage of the initial number of bound cells that remain attached at the indicated flow rates. (B) The percentage of tethering, rolling, and firmly adhered cells at a 1-dyn/cm2 flow rate. Figure shows an example of the effect of a test compound (PTx) on the interaction of CXCL12-induced Molt4 cells with fibronectin. PTx diminishes cell attachment to FN at low flow rates (right panel). Using this protocol, it can be observed that PTx differentially affects the distinct features of cell adhesion; it does not alter the percentage of rolling cells, but modifies the percentage of tethering and stably arrested cells (right panel).
    View Image
  •  FigureFigure 12.11.5 Transwell insert for the evaluation of cell migration and invasion (Basic Protocols 2 and 3).
    View Image
  •  FigureFigure 12.11.6 CCL2-induced migration of MonoMac-1 cells. Cells (5 × 105/well) were placed on 5-µm transwells and allowed to migrate for 2 hr in response to different concentrations of CCL2. The figure shows the bell-shaped concentration curve (filled circles) and the theoretical effect of blocking CCL2 responses using an antagonist compound (filled squares). Arrows show that blocking the response to 1 nM CCL2 completely abrogates the response, while blocking the activity of 50 nM CCL2 increases cell migration. In contrast, the use of pertussis toxin (PTx), which blocks CCL2-induced signaling pathways, completely blocks responses to the chemokine at all concentrations tested (open circles).
    View Image
  •  FigureFigure 12.11.7 Chemotaxis of CCR2/CCR5 HEK 293 cells was assessed in 8-µm transwells, as described in Basic Protocol 2, using various concentrations of CCL2, CCR2-01, CCR2-02, or CCR2-04. To block CCL2-mediated chemotaxis, CCR2-01 or CCR2-04 (10 µg/ml) were added to the lower well simultaneously with the chemokines. Chemotaxis in response to CCL5 (10 nM) or CXCL12 (40 nM), and the effect of CCR2-01 (100 µg/ml), were assessed in a similar manner. Data represent the mean ± SD of triplicate determinations. Figure shows the analysis of test compounds following this protocol. The antagonist activity of a test compound (CCR2-04) is determined by its capacity to block CCL2-induced chemotaxis (left panel). The agonist behavior of a test compound (CCR2-02) can be analyzed by its ability to promote cell migration (central panel). A control test compound (CCR2-01) has no effect on blocking or promoting cell migration. Reprinted with permission from Rodríguez-Frade et al. (2008).
    View Image
  •  FigureFigure 12.11.8 CCR2-05 MAb blocks CCL2-mediated responses in cynomolgus monkey peripheral blood mononuclear cells (PBMCs). Cells were incubated with the indicated concentrations of CCR2-05 and allowed to migrate in response to CCL2. Cells that migrated to the lower chamber were counted and the results expressed as a migration index. Data show mean ± SD of triplicate determinations. Reprinted with permission from Mellado et al. (2007).
    View Image
  •  FigureFigure 12.11.9 Protocol used for determination of adherent cell migration using a 96-well transmigration chamber (Alternate Protocol 3).
    View Image
  •  FigureFigure 12.11.10 CXCL12-induced migration of HEK-293 cells to fibronectin. Untreated cells or cells treated with DMSO, pertussis toxin (PTx), or cholera toxin (CTx) were placed on 10-µm frame filters and allowed to migrate for 6 hr in a Neuro Probe migration chamber in response to different CXCL12 concentrations. (A) Filters were stained with crystal violet and scanned. (B) Cells on the filter were quantified by densitometry of stained spots using Image J software, with the results expressed in arbitrary units.
    View Image
  •  FigureFigure 12.11.11 Synthetic peptides containing Ile52 and Val150 block CCR5-induced responses. Upper panels: CCR5wt-transfected L1.2 cells (left) or hPBL from healthy donors (right) were treated with R5wtpep or R5mutpep peptides and allowed to migrate in response to CCL5. Cells that migrated to the lower chamber were counted and expressed as a migration index. Data represent the mean ± SD of triplicate determinations. Lower panels: hPBL from healthy donors were labeled with Cell Tracker orange or green and treated with R5wtpep (green, represented here by darker gray; top bars), R5mutpep (orange, represented here by lighter gray; lower bars) or PBS (green and orange), injected intravenously into mice, and their accumulation in spleen analyzed after intrasplenic injection of CCL5. Results of three independent experiments are expressed as the migration index of CCR5+ cells (mean ± SD) calculated as indicated in Figure 12.11.7. The figure also shows the orange/green-stained cell ratio for each condition. Reprinted with permission from Hernanz-Falcón et al. (2004).
    View Image

Videos

Literature Cited

Literature Cited
    Donovan, J. and Brown, P. 1998. Anesthesia. Curr. Protoc. Immunol. 27: 1.4.1-1.4.5.
    Donovan, J. and Brown, P. 2006a. Parenteral injections. Curr. Protoc. Immunol. 73: 1.6.1-1.6.10.
    Donovan, J. and Brown, P. 2006b. Euthanasia. Curr. Protoc. Immunol. 73: 1.8.1-1.8.4.
    Frade, J.M., Mellado, M., del Real, G., Gutierrez-Ramos, J.C., Lind, P., and Martinez, A.C. 1997. Characterization of the CCR2 chemokine receptor: Functional CCR2 receptor expression in B cells. J. Immunol. 159: 5576-5584.
    Grabovsky, V., Feigelson, S., Chen, C., Bleijs, D.A. Peled, A., Cinamon, G., Baleux, F., Arenzana-Seisdedos, F., Lapidot, T., van Kooyk, Y., Lobb, R.R., and Alon, R. 2000. Subsecond induction of alpha4 integrin clustering by immobilized chemokines stimulates leukocyte tethering and rolling on endothelial vascular cell adhesion molecule 1 under flow conditions. J. Exp. Med. 192: 495-506.
    Hernanz-Falcon, P., Rodríguez-Frade, J.M., Serrano, A., Juan, D., del Sol, A., Soriano, S.F., Roncal, F., Gomez, L., Valencia, A., Martinez, A.C., and Mellado, M. 2004. Identification of amino acid residues crucial for chemokine receptor dimerization. Nat. Immunol. 5: 216-223.
    Ley, K., Laudanna, C., Cybulsky, M.I., and Nourhargh, S. 2007. Getting to the site of inflammation: The leukocyte adhesion cascade updated. Nat. Rev. Immunol. 7: 678-689.
    Mellado, M., Rodríguez-Frade, J.M., Manes, S., and Martinez-A., C. 2001. Chemokine signaling and functional responses: The role of receptor dimerization and TK pathway activation. Annu. Rev. Immunol. 19: 397-421.
    Mellado, M., Martin de Ana, A., Gomez, L., Martinez-A, C., and Rodríguez-Frade, J.M. 2007. Chemokine receptor 2 blockade prevents asthma in a cynomolgus monkey model. J. Pharmacol. Exp. Ther. 324: 769-775.
    Pachynski, R.K., Wu, S.W., Gunn, M.D., and Erle, D.J. 1998. Secondary lymphoid-tissue chemokine (SLC) stimulates integrin alpha 4 beta 7-mediated adhesion of lymphocytes to mucosal addressin cell adhesion molecule-1 (MAdCAM-1) under flow. J. Immunol. 161: 563-571.
    Reeves, J.P., Reeves, P.A., Chin, L.T. 1991. Survival surgery: Removal of the spleen or thymus. Curr. Protoc. Immunol. 2: 1.10.1-1.10.11.
    Robinson, J.P., Darzynkiewicz, Z., Hoffman, R., Nolan, J.P., Orfao, A., Rabinovitch, P.S., and Watkins, S. 2008. Current Protocols in Cytometry. John Wiley & Sons, Hoboken, N.J.
    Rodríguez-Frade, J.M., del Real, G., Serrano, A., Hernanz-Falcón, P., Soriano, S.F., Vila-Coro, A.J., Martín de Ana, A:, Lucas, P., Prieto, I., Martínez-A., C., and Mellado, M. 2004. Blocking HIV-1 infection via CCR5 and CXCR4 receptors by acting in trans on the CCR2 chemokine receptor EMBO J. 23: 66-76.
    Sahai, E. 2007. Illuminating the metastatic process. Nat. Rev. Cancer 7: 737-749.
    Sanz-Rodriguez, F., Hidalgo, A., and Teixido, J. 2001. Chemokine stromal cell-derived factor-1alpha modulates VLA-4 integrin-mediated multiple myeloma cell adhesion to CS-1/fibronectin and VCAM-1. Blood 97: 346-351.
    Springer, T.A. 1994. Traffic signals for lymphocyte recirculation and leukocyte emigration: The multistep paradigm. Cell 76: 301-314.
    Stein, J.V., Soriano, S.F., M'Rini, C., Nombela-Arrieta, C., de Buitrago, G.G., Rodríguez-Frade, J.M., Mellado, M., Girard, J.P., and Martinez-A, C. 2003. CCR7-mediated physiological lymphocyte homing involves activation of a tyrosine kinase pathway. Blood 101: 38-44.
    Ulbrich, H., Eriksson, E.E., and Lindbom, L. 2003. Leukocyte and endothelial cell adhesion molecules as targets for therapeutic interventions in inflammatory disease. Trends Pharmacol. Sci. 24: 640-647.
    Vicente-Manzanares, M., and Sanchez-Madrid, F. 2004. Role of the cytoskeleton during leukocyte responses. Nat. Rev. Immunol. 4: 110-122.
    Yamaguchi, H., Wyckoff, J., and Condeelis, J. 2005. Cell migration in tumors. Curr. Opin. Cell Biol. 17: 559-564.
 Internet Resources
    http://www.ramicweb.com

Web page of the Spanish Cell Adhesion and Migration Network (in English).

    http://bme.virginia.edu/ley/

Page from the University of Virginia, with data on the molecules involved in the leukocyte adhesion cascade.

    http://apresslp.gvpi.net/apcyto/lpext.dll?f=templates&fn=main-h.htm&2.0

Cytokine Reference. An on-line book with detailed information on cytokines, chemokines, growth factors, and other host defense mediators. Information organized by family, ligand, receptor, disease or cell type.

    http://www.neuro.wustl.edu/neuromuscular/lab/adhesion.htm#

Detailed information on molecules involved in cell movement: selectins, integrins, immunoglobulin superfamily, and cadherins.

    http://www.cellmigration.org/index.shtml

Cell Migration Gateway: a comprehensive and regularly updated resource for anyone interested in cell migration. Collaboration between the Cell Migration Consortium and Nature Publishing Group.

GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library